Phosphorescence is the result of a three-stage process. In the first stage, energy is supplied by an external source, such as an incandescent lamp or a laser, and absorbed by the phosphorescent compound, creating excited electronic triplet states (as opposed to fluorescence, which only has a singlet excited state). In the second stage, the excited states exist for a finite time during which the phosphorescent compound undergoes conformational changes and is also subject to a multitude of possible interactions with its molecular environment. During this time, the energy of the excited states is partially dissipated, yielding relaxed states from which phosphorescence emission originates. The third stage is the phosphorescence emission stage wherein energy is emitted, returning the phosphorescence compound to its ground states. The emitted energy is lower than its excitation energy (light or laser) and thus of a longer wavelength. This shift or difference in energy or wavelength allows the emission energy to be detected and isolated from the excitation energy.
Various phosphorescent compounds, such as metalloporphyrins, have been proposed for use in immunoassays. Unfortunately, many of the proposed techniques fail to solve the problem of quenching. Specifically, oxygen and water are strong quenchers of triplet states and may cause decay of the phosphorescence signal, thereby limiting its use in most practical assay applications. In addition, many of the techniques that have been proposed are simply ill equipped for use in lateral flow, membrane-based devices. For example, in a lateral flow, membrane-based assay device, the concentration of the analyte is reduced because it is diluted by a liquid that may flow through the porous membrane. However, background interference becomes increasingly problematic at such low analyte concentrations because the phosphorescent intensity is relatively low. Because the structure of the membrane also tends to reflect the excited light, the ability of a detector to accurately measure the phosphorescent intensity of the labeled analyte is substantially reduced. In fact, the intensity of the emitted phosphorescence signal may be three to four orders of magnitude smaller than the excitation light reflected by the porous membrane. Many membranes, such as nitrocellulose membranes, also exhibit strong fluorescence when excited in the UV and visible regions. This fluorescence can interfere with the accuracy of phosphorescence measurements.
As such, a need currently exists for a simple, inexpensive, and effective system for using phosphorescence as a detection technique for membrane-based, lateral flow assay devices.